The thymus generates a vast excess of cells that fail to complete maturation and die in situ. This is a result of the processes of beta-selection, positive selection, and negative selection that place stringent controls on various stages of T cell development. Both beta-selection and positive selection are cell differentiation events that result in stable changes in gene expression. There is still much to learn about the molecular control of gene expression during these critical developmental transitions. We have recently identified a gene encoding a novel regulator of thymocyte selection we termed TOX (thymocyte selection associated HMG-box protein). As its name implies, TOX is a member of the HMG-box superfamily of nuclear proteins. HMG-box proteins are architectural factors that bind DNA and regulate formation of mutiprotein transcriptional regulatory complexes and/or are involved in regulating chromatin accessibility. TOX also belongs to a small conserved subfamily of HMG-box proteins. Expression of TOX in the thymus is tightly regulated and is associated with both beta-selection and positive selection. Data indicate that expression of TOX in the thymus of transgenic mice is sufficient to initiate both of these differentiation processes. This includes promotion of CD8 T cell lineage commitment at the expense of CD4 lineage commitment. We propose to further investigate the biological functions of this novel nuclear protein. Specific approaches are outlined to; determine the expression of TOX and family members during lymphocyte development, analyze biochemical characteristics of TOX, determine subcellular and intranuclear localization of the protein, identify signaling pathways that regulate its expression, perform a structure/function analysis of various forms of TOX in vitro and in vivo, produce TOX deficient mice, identify other proteins that interact with domains of TOX and identify gene targets for this nuclear protein in the context of positive selection.